Microfluidic Polyacrylamide Gel Electrophoresis with in Situ

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Anal. Chem. 2009, 81, 8177–8184

Microfluidic Polyacrylamide Gel Electrophoresis with in Situ Immunoblotting for Native Protein Analysis Mei He and Amy E. Herr* Department of Bioengineering, University of California, Berkeley, California 94720 We introduce an automated immunoblotting method that reports protein electrophoretic mobility and identity in a single streamlined microfluidic assay. Native polyacrylamide gel electrophoresis (PAGE) was integrated with subsequent in situ immunoblotting. Integration of three PA gel elements into a glass microfluidic chip achieved multiple functions, including (1) rapid protein separation via on-chip PAGE, (2) directed electrophoretic transfer of resolved protein peaks to an in-line blotting membrane, and (3) high-efficiency identification of the transferred proteins using antibody-functionalized blotting membranes. In-chip blotting membranes were photopatterned with biotinylated antibody using streptavidin polyacrylamide (PA) thus yielding postseparation sample analysis. No pressure driven flow or fluid valving was required, as the assay was operated by electrokinetically programmed control. A model sample of fluorescently labeled BSA (negative control), r-actinin, and prostate specific antigen (PSA) was selected to develop and characterize the assay. A 5 min assay time was required without operator intervention. Optimization of the blotting membrane (geometry, operation, and composition) yielded a detection limit of ∼0.05 pg (r-actinin peak). An important additional blotting fabrication strategy was developed and characterized to allow vanishingly small antibody consumption (∼1 µg), as well as end-user customization of the blotting membrane after device fabrication and storage. This first report of rapid on-chip protein PAGE integrated with in situ immunoblotting forms the basis for a sensitive, automated approach applicable to numerous forms of immunoblotting. Immunoaffinity methods are essential to clinical diagnostics, as well as fundamental life science investigations. Among immunoaffinity methods, both immunoassays and immunoblotting approaches are workhorse techniques used for decades to detect specific analytes in complex biological fluids.1-4 Whereas immu* To whom correspondence should be addressed. Phone: (510) 666-3396. Fax: (510) 642-5835. E-mail: [email protected]. (1) Gershoni, J. M.; Palade, G. E. Anal. Biochem. 1983, 131, 1–15. (2) Anderson, D. J.; Lente, F. V.; Apple, F. S.; Kazmierczak, S. C.; Lott, J. A.; Gupta, M. K.; McBride, N.; Katzin, W. E.; Scott, R. E.; Toffaletti, J.; Menendez-Botet, C. J.; Schwartz, M. K.; Castellani, W. J.; Hage, D. S.; Allen, R. C.; Griffiths, J. C.; Hepler, B. R.; Touchstone, J. C.; Skogerboe, K. J.; Wang, J.; Kuesel, A. C.; Kroft, T.; Smith, I. C. P.; Haas, R. G.; Chou, D. Anal. Chem. 1991, 63, 165R–270R. 10.1021/ac901392u CCC: $40.75  2009 American Chemical Society Published on Web 09/04/2009

noassays report the presence of protein using immunorecognition, immunoblotting reports both apparent mobility (i.e., protein size, charge-to-mass ratio) and antigen-antibody interaction. Immunoblotting comprises a suite of assays including: Western and Far Western blotting for protein presence and protein-protein interaction, Northern blotting for RNA, and Southern blotting for DNA. To obtain both mobility and immunorecognition characteristics for proteins, slab polyacrylamide gel electrophoresis (PAGE) is typically coupled with “blotting” of separated proteins on a membrane. Subsequently, blotted proteins are probed with reporter antibodies and detected via various stains including chemiluminescence.5 Information about the mobility of the protein from PAGE and the specificity of the antibody-antigen interaction enables a target protein to be identified in the midst of a complex protein mixture. Immunoblotting of proteins is routinely used in basic biological research studies, as well as for confirmatory clinical diagnostic assays (i.e., HIV and Lyme disease). While a powerful assay platform, one major disadvantage of conventional immunoblotting (i.e., slab-gel PAGE and subsequent manual “blotting”) is the requirement for tedious labor-intensive and timeintensive manual intervention at various steps of the assay.6 Surprisingly, immunoblotting formats have changed little since first introduction by Towbin in 1979.7 Recently, automation of immunoblotting has been reported using a capillary, not slab-gel, format.8,9 Capillary isoelectric focusing (IEF) was used to separate proteins, including protein isoforms.10 The capillary IEF separation was followed by UV surface-immobilization of resolved proteins on a photoactivatable capillary surface. Subsequent detection was accomplished by flushing out all nonsurface cross-linked materials and flushing in (3) Tissot, J. D.; Vu, D. H.; Aubert, V.; Schneider, P.; Vuadens, F.; Crettaz, D.; Duchosal, M. A. Proteomics 2002, 2, 813–824. (4) Righetti, P. G.; Castagna, A.; Antonucci, F.; Piubelli, C.; Cecconi, D.; Campostrini, N.; Rustichelli, C.; Antonioli, P.; Zanusso, G.; Monaco, S.; Lomas, L.; Boschetti, E. Clin. Chim. Acta 2005, 357, 123–139. (5) Sun, L.; Ghosh, I.; Barchevsky, T.; Kochinyan, S.; Xu, M. Q. Methods 2007, 42, 220–226. (6) Wu, Y.; Li, Q.; Chen, X. Z. Nat. Protoc. 2007, 2, 3278–3284. (7) Towbin, H.; Staehelin, T.; Gordon, J. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 4350–4354. (8) Guzman, N. A.; Park, S. S.; Schaufelberger, D.; Hernandez, L.; Paez, X.; Rada, P.; Tomlinson, A. J.; Naylor, S. J. Chromatogr., B 1997, 697, 37–66. (9) Peoples, M. C.; Phillips, T. M.; Karnes, H. T. J. Pharm. Biomed. 2008, 48, 376–382. ´ Neill, R. A.; Bhamidipati, A.; Bi, X.; Deb-Basu, A.; Cahill, L.; Ferrante, J.; (10) O Gentalen, E.; Glazer, M.; Gossett, J.; Hacker, K.; Kirby, C.; Knittle, J.; Loder, R.; Mastroieni, C.; MacLaren, M.; Mills, T.; Nguyen, U.; Parker, N.; Rice, A.; Roach, D.; Suich, D.; Voehringer, D.; Voss, K.; Yang, J.; Yang, T.; Horn, P. B. V. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 16153–16158.

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reagents for antibody-based identification with chemiluminescence.11,12 A robotic interface was required to integrate fluid exchange steps with the capillary tube geometry (i.e., single inlet, single outlet) used. One difficulty in integration of multiassay steps in capillary systems arises from a need for external fixturing to multiple reservoirs and interfaces. In a like-minded effort to advance the ease and speed of immunoblotting, we note that unified, integrated design is a hallmark of microsystems. Integration of multiple functions in a single microfluidic system substantially benefits automation of multistage assays through facile onchip interfacing.13 Dexterity in sample manipulation and the ability to fabricate interconnecting channels with zero dead volume contribute to high performance and low sample loss. Additional compelling advantages of microfluidic systems include reduced manual intervention, nominal sample consumption, rapid results, improved assay precision, and digital data collection essential for archiving and comparison.14-16 Coupled with automated fluid manipulation, the aforementioned factors make microfluidic devices exceptionally well-suited formats for multistep analyses.17-19 An area of notable recent growth in reports of multistep assays includes on-chip integration of sample handling and pretreatment with electrophoretic protein analysis, an area relevant to the study reported here. Examples of the “upstream” integration of function with assays include sample concentrators,20 mixers,21 and microreactors.22 These versatile preparatory functions can greatly enhance the final assay performance and yield automated, multifunctional protein measurement tools.23-27 In this article, we introduce a new approach to immunoblotting that takes advantage of microfluidic technology to simplify integration and, hence, multistep assay operation. Further, and most importantly, while tremendous enhanced functionality has been gained from “upstream” sample preparation using integrated polymer features, we focus here on enhanced postseparation (11) Knittle, J. E.; Roach, D.; Horn, P. B. V.; Voss, K. O. Anal. Chem. 2007, 79, 9478–9483. ´ Neill, (12) Fan, A. C.; Deb-Basu, D.; Orban, M. W.; Gotlib, J. R.; Natkunam, Y.; O R.; Padua, R. A.; Xu, L.; Taketa, D.; Shirer, A. E.; Beer, S.; Yee, A. X.; Voehringer, D. W.; Felsher, D. W. Nat. Med. 2009, 15, 566–571. (13) Hou, C.; Herr, A. E. Electrophoresis 2008, 29, 3306–3319. (14) Mauk, M. G.; Ziober, B. L.; Chen, Z.; Thompson, J. A.; Bau, H. H. Ann. N.Y. Acad. Sci. 2007, 1098, 467–475. (15) West, J.; Becker, M.; Tombrink, S.; Manz, A. Anal. Chem. 2008, 80, 4403– 4419. (16) Peoples, M. C.; Karnes, H. T. J. Chromatogr., B 2008, 866, 14–25. (17) Yeung, S. H.; Liu, P.; Bueno, N. D.; Greenspoon, S. A.; Mathies, R. A. Anal. Chem. 2009, 81, 210–217. (18) Meagher, R. J.; Hatch, A. V.; Renzi, R. F.; Singh, A. K. Lab Chip 2008, 8, 2046–2053. (19) Fan, R.; Vermesh, O.; Srivastava, A.; Yen, B. K. H.; Qin, L.; Ahmad, H.; Kwong, G. A.; Liu, C. C.; Gould, J.; Hood, L.; Heath, J. R. Nat. Biotechnol. 2008, 26, 1373–1378. (20) Hatch, A. V.; Herr, A. E.; Throckmorton, D. J.; Brennan, J. S.; Singh, A. K. Anal. Chem. 2006, 78, 4976–4984. (21) Johnson, T. J.; Ross, D.; Locascio, L. E. Anal. Chem. 2002, 74, 45–51. (22) Kawabata, T.; Wada, H. G.; Watanabe, M.; Satomura, S. Electrophoresis 2008, 29, 1399–1406. (23) Phillips, T. M.; Wellner, E. F. Electrophoresis 2007, 28, 3041–3048. (24) Srivastava, N.; Brennan, J. S.; Renzi, R. F.; Wu, M.; Branda, S. S.; Singh, A. K.; Herr, A. E. Anal. Chem. 2009, 81, 3261–3269. (25) Herr, A. E.; Hatch, A. V.; Throckmorton, D. J.; Tran, H. M.; Brennan, J. S.; Giannobile, W. V.; Singh, A. K. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 5268–5273. (26) Hu, G.; Gao, Y.; Sherman, P. M.; Li, D. Microfluid. Nanofluid. 2005, 1, 346–355. (27) Clark, M. A.; Sousa, K. M.; Jennings, C.; MacDougald, O. A.; Kennedy, R. T. Anal. Chem. 2009, 81, 2350–2356.

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or “downstream” functionality enabled through functional polymer features. Here we integrate sample analysis (PAGE) with postseparation affinity-based protein blotting in an effort to develop a “hands-free” microanalytical immunoblot. The reported PAGE in situ immunoblotting technology relies on multiple, functionalized polyacrylamide (PA) gels photopatterned in specific regions of a planar, glass microfluidic device. This assay integrates rapid, high-resolution native PAGE with antibody-functionalized PA blotting membranes to yield in situ reporting of protein mobility and antibody-affinity. After PAGE, electrophoretic transfer of resolved species to a blotting membrane is demonstrated as a directed, efficient method for protein identification, without a need for pressure-driven flow and valving. Using the PAGE in situ immunoblot, we demonstrate limited consumption of expensive detection reagents (i.e., antibodies) and precious starting samples, as expected from microfluidic formats.14 Postseparation blotting takes advantage of the high surface areato-volume ratio and fast mass transport available in said geometries, which leads to a significant decrease in analysis time (from hours to seconds).15 The platform technology introduced forms a promising basis for our development of quantitative, automated microfluidic immunoblotting. MATERIALS AND METHODS Reagents. The water-soluble photoinitiator 2,2-azobis[2-methylN-(2-hydroxyethyl) propionamide] (VA-086) was purchased from Wako Chemicals (Richmond, VA). 3-(Trimethoxysilyl)-propyl methacrylate (98%), perchloric acid (70%, ACS grade), hydrogen peroxide (30%, ACS grade), glacial acetic acid (ACS grade), methanol (ACS grade), and 30% (29:1) acrylamide/bis-acrylamide were purchased from Sigma. Streptavidin-acrylamide (SA) was purchased from Invitrogen (Carlsbad, CA). Premixed 10× Trisglycine native electrophoresis buffer (25 mM Tris, pH 8.3, 192 mM glycine) was purchased from Bio-Rad (Hercules, CA). Alexa Fluor 488 conjugated bovine serum albumin (BSA) and FITCbiotin were used as negative and positive controls, respectively (Sigma Aldrich, St. Louis, MO). R-Actinin and biotin conjugated anti-actinin were purchased from Cytoskeleton, Inc. (Denver, CO). Free prostate specific antigen (PSA) and biotinylated monoclonal anti-PSA were purchased from EXBIO (Praha, Czech Republic). The proteins R-actinin and PSA were fluorescently labeled in-house using Alexa Fluor 488 protein labeling kits per the supplier’s instructions (Life Technologies, Carlsbad, CA) and purified by P-6 Bio-Gel columns (Bio-Rad, Hercules, CA). Labeled proteins were stored at 4 °C in the dark until use. Chip Fabrication. Glass microfluidic chips were designed inhouse and fabricated using standard wet etch processes by Caliper Life Sciences (Hopkinton, MA). The sample (S), sample waste (SW), buffer (B, B1, B2), and buffer waste (BW) reservoirs are indicated in Figure 1a. Chip layouts consisted of a double-T junction having a 2.5 mm separation channel connected to a second junction needed for patterning of the downstream blotting membranes. Channels were ∼15 µm deep and ∼80 µm wide. An initial sample volume of 5 µL was employed. Channel surfaces were first functionalized for covalent linkage to PA gel using a 2:3:2:3 ratio mixture of 3-(trimethoxysilyl) propyl methacrylate, glacial acetic acid, deionized water, and methanol. After a 20 min static incubation, methanol was flushed through all channels for 30 min followed by a drying nitrogen purge.

Figure 1. Fabrication of microfluidic native PAGE in situ immunoblotting device: (a) schematic of chip layout (not to scale). Fluid reservoirs are labeled according to contents: S, sample; B, buffer; SW, sample waste; BW, buffer waste. Polyacrylamide gel composition is indicated by grayscale (% T and % C are percentage of total acrylamide and cross-linker, respectively). The inset images show a 10× view of a streptavidin functionalized blotting membrane photopatterned within the channel. (b) Schematic depicting fabrication steps for blotting membrane: one-step prepatterning strategy and two-step custom patterning strategy. In the custom patterning strategy, loading of the biotinylated antibody is via applied electric current (indicated by i).

Fabrication of Blotting Membranes. Chrome mask-based lithography via a UV objective (UPLANS-APO 4×, Olympus) microscope system (IX-70, Olympus, Melville, NY) was used to fabricate the blotting membranes. A mercury bulb was used as the excitation source (330-375 nm) and mask alignment to the chip was performed using a manual adjust x-y translation stage.28 For photopatterning, a 5 min UV excitation was used to form the blotting membranes (8% T, including SA). To yield immunoaffinity-based protein detection on the blotting membrane, SA covalently linked to the PA gel matrix was used to immobilize biotinylated antibodies. As illustrated in Figure 1b, antibody immobilization in the PA blotting membranes was conducted by one of two methods, a prepatterning strategy or a custom patterning strategy. The single-step prepatterning strategy consisted of a concurrent photopatterning and antibody immobilization step that employs PA gel precursor solutions that contain both SA and biotinylated antibody. The binding capacity of the streptavidin blotting membrane was demonstrated by introducing FITC-biotin (Figure 1a, inset). The alternate, two-step custom patterning strategy consisted of (i) photopatterning of a gel precursor solution containing SA, followed by (ii) antibody (28) Das, C.; Zhang, J.; Denslow, N. D.; Fan, Z. H. Lab Chip 2007, 7, 1806– 1812.

immobilization that utilized a slow electrophoretic introduction of biotinylated antibody through the streptavidin-decorated PA gel membrane (i.e., 10 V/cm electrophoresis from well B2, Figure 1a). The custom patterning strategy resulted in the immobilization of biotinylated antibodies after fabrication and storage, making customization possible by the end-user. Characterization of the custom patterning strategy for a range of patterning times suggests that exposed streptavidin binding sites in the blotting membrane are sufficiently occupied after a 10 min antibody patterning step (10 V/cm, 1 µM anti-actinin). Fabrication of Loading and Separation Gels. After patterning of the blotting membrane, all unpolymerized open channels were flushed with buffer for 1 min using vacuum in preparation for fabrication of the loading and separation gels. The chip was then soaked in a buffer solution to allow diffusive dilution of unpolymerized precursor between the blotting membrane and reservoir BW, although complete removal of the unpolymerized precursor was not necessary. PA gels of various acrylamide concentrations were photopatterned in the microdevices in a manner similar to our previous reports.20,29,30 Briefly, degassed PA gel precursor solution for the separation gel (8% T) was wicked or gently pressure-filled (via syringe) into the channels. After all channels were loaded, high viscosity 5% 2-hydroxyethyl cellulose (HEC, Sigma, average MW ∼720 000) drops were gently applied onto each fluid-containing reservoir. The 5% HEC solution quickly eliminated hydrostatic flow resulting in quiescent fluid conditions inside the channels, as is needed for high-resolution photopolymerization (1 min photopatterning via the UV objective setup). After polymerization of the separation channel, the HEC solution on each reservoir was flushed off by buffer solution. Then, a larger pore-size sample loading gel was formed using 3% T acrylamide solution and an 8 min flood exposure of the chip to a filtered mercury lamp (300-380 nm) located 15 cm away (100 W, UVP B100-AP, Upland, CA) with cooling fan. The photopolymerization times reported were determined empirically based on the intensity of each UV light source, composition of acrylamide precursor solution, and desired pore-size to achieve optimal gel performance for the desired function. PA gels were visually inspected and typically showed a well-defined, uniform blotting structure with sharp interfaces and channels free of bubbles and voids (Figure 1a, inset). Chip Reuse. After use, the glass chips were routinely regenerated through removal of the cross-linked PA gels that form the loading and separation gels, as well as the blotting membranes. An empirically determined regeneration protocol consisted of soaking used chips (containing PA gels) in a 2:1 ratio solution of perchloric acid and hydrogen peroxide at 75 °C overnight. After soaking, channels were flushed with 0.1 M sodium hydroxide for 30 min. Visual inspection showed negligible residual PA gel inside the channels after treatment. Subsequent fabrication of crosslinked PA gels in the recycled glass chips yielded >90% success, which is on par with multistep photopatterning device yields in new glass chips. Apparatus and Imaging. Electrophoretic transport was used to mobilize species through the gel elements for all steps of the (29) Herr, A. E.; Throckmorton, D. J.; Davenport, A. A.; Singh, A. K. Anal. Chem. 2005, 77, 585–590. (30) Lo, C. T.; Throckmorton, D. J.; Singh, A. K.; Herr, A. E. Lab Chip 2008, 8, 1273–1279.

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PAGE in situ immunoblot. After sample introduction, assay operation was programmable and controlled via a high voltage power supply equipped with platinum electrodes (Labsmith HVS448, Livermore, CA). Samples were loaded by applying a +800 V potential at the SW reservoir and grounding the S reservoir for ∼2 min. During loading, both the B and BW reservoirs were grounded to form a well-defined “pinched” injection plug.31 For separation, various potentials were applied at the BW reservoir while grounding the B reservoir with a push-back voltage at reservoir S and SW.29 To ensure minimal cross-contamination between samples and reproducible runs, all channels were electrophoretically flushed with buffer every other run. Images were collected using an inverted epi-fluorescence microscope (IX70, Olympus, Melville, NY) equipped with a 10× objective (NA 0.3), filter cube optimized for GFP detection, and an x-y translation stage. A 1392 × 1040 Peltier-cooled interline CCD camera (CoolSNAP HQ2, Roper Scientific, Trenton NJ) was employed to monitor protein migration and blotting. Unless otherwise stated, the CCD exposure time was 400 ms. Electropherograms were generated by measuring fluorescence intensity (FL signal) in a detection region of interest (ROI) in the captured CCD image time sequences. The ROI occupied a small region of the channel (∼10 µm wide and 80 µm long), and intensity values were extracted from the ROI using ImageJ. Nonlinear least-squares fitting of the signal was performed using data analysis and graphing software (OriginPro 8.0, Northampton, MA). RESULTS AND DISCUSSION Microfluidic PAGE in Situ Immunoblot Assay. The native PAGE in situ immunoblot consists of three photopatterned PA gel regions in one glass device (Figure 1a). Each region is optimized for a specific function, namely, sample loading, protein separation, transfer, and in situ immunoblotting. The assay reports both apparent electrophoretic protein mobility and protein identity through immunodetection. To yield these two pieces of information, the assay proceeds in a electrokinetically controlled three-step sequence: (1) sample is loaded into the chip through a large pore-size “loading” gel, (2) native proteins are electrophoretically injected and separated through a smaller pore-size separation gel matrix, and (3) proteins are subsequently electrophoretically transported through an antibodyfunctionalized blotting membrane. During the separation step, protein species are resolved based on differential migration through the gel matrix. As the PAGE demonstrated here is under native conditions, species resolve owing to differences in both charge-to-mass ratio and the overall size of the protein. After the PAGE separation, electrophoresis is used to transport resolved proteins to and through the contiguous blotting membrane which has a composition similar to the separation matrix albeit with immobilized antibodies present. As illustrated schematically in Figure 2a, proteins lacking specific affinity for the immobilized antibody migrate through the blotting membrane, while proteins having specific affinity for the immobilized antibody can be retained on the blotting membrane. For characterization of the blotting capture efficiency, electropherograms were collected both upstream and (31) He, M.; Zeng, Y.; Sun, X.; Harrison, D. J. Electrophoresis 2008, 29, 2980– 2986.

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Figure 2. Operation of microfluidic native PAGE in situ immunoblotting assay: (a) schematic depicting assay operation. A native protein sample is electrophoretically separated and electrophoretically transferred to a contiguous antibody-functionalized blotting membrane, and specific target proteins are identified by interaction with antibodies immobilized in the blotting membranes. (b) Timesequence of micrographs show native PAGE analysis of BSA (left panel) and R-actinin (right panel) with subsequent in situ immunoblotting. The blotting membrane is marked with an asterisk (*). R-Actinin was blotted to membrane (+) with negligible capture of negative control BSA (-). (c) Electropherograms collected from upstream (left panel) and downstream (right panel) of the blotting membrane in (b) show selective extraction of the target protein, R-actinin (slowest peak). Unmarked peak is attributed to an impurity. Applied electric field is 100 V/cm.

downstream of the blotting membrane. The specificity and the selectivity of the blotting membrane provided the capability to capture target protein, which was demonstrated by in situ immunoblotting of a mixture of proteins integrated with native PAGE analysis. Characterization results of biotinylated antiactinin decorated blotting membranes (Figure 2b,c) reveal appreciable resolution (R ) 7.1) between the two model species

Figure 3. Microfluidic native PAGE in situ immunoblotting is a high specificity assay: (a) protein signal on blotting membranes allows assessment of blotting specificity. Inverted grayscale CCD fluorescence images show fluorescence of proteins bound to inchannel antibody-functionalized blotting membranes. Each analysis considered a single protein challenge to each blotting membrane configuration through a 60 s loading duration at 300 V/cm. The protein concentration was ∼0.2 µM in each case.

matched antibody-antigen pairs) was ∼90% (Figure 3), which indicates significant binding compared to the off-axis capture (i.e., nonmatched antibody-antigen pairs). The results indicate little to no discernible cross-reactivity or nonspecific adsorption for the proteins and antibodies considered. For native PAGE in situ immunoblotting of more complex mixtures or different species than those considered here, further assessment of cross-reactivity of specific antibodies will be necessary, as is commonly done in protein microarrays, ELISAs, and slab gel immunoblotting. Our initial characterization results support the use of biotinylated antibodies immobilized in PA gels (containing SA) as a means to specifically identify antigens. Blotting Membrane Capture Efficiency. As a metric of blotting membrane performance, capture efficiency (capture %) was defined as

capture % ) within a 30 s native PAGE analysis time. Electropherograms collected upstream and downstream of the blotting membrane suggest selective blotting of R-actinin with negligible capture of BSA (negative control), as observed in Figure 2b,c. The measured apparent mobilities for R-actinin and BSA were 1.07 × 10-4 and 1.57 × 10-4 cm2 V-1 s-1, respectively. In the sample analysis presented here, the time required for native PAGE in situ immunoblotting was less than 5 min, including sample loading ∼2 min, separation 80%) when the blotting membrane precursor contains at least a 4 µM streptavidinacrylamide (SA). For blotting membranes fabricated with 2 µM biotinylated anti-actinin present without SA in the precursor (Table 1, strategy A), an 8.6% capture efficiency (RSD ) 4.1%, n ) 5) was observed for R-actinin. The detection of R-actinin even when no SA was present may be attributable to physical copolymerization of large molecular weight biotinylated antiactinin in the cross-linked PA gel. Nevertheless, the results reported here suggest incorporation of SA in the PA gel precursor solution yields specific attachment of biotinylated antibodies as immuno-recognition sites. When no SA or biotinylated antibody was present in the blotting membrane, no significant binding of R-actinin was observed. Analytical Chemistry, Vol. 81, No. 19, October 1, 2009

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Table 1. Blotting Membrane Capture Efficiency: Comparison between Prepatterned and Custom-Patterned Antibody Immobilization Strategiesa strategy A (prepatterning) b

[streptavidin-acrylamide, SA] ratio of SA to biotin anti-actinin average capture % of R-actinin ± RSD % (n ) 5) a

0 µM 0:2 8.6 ± 4.1

0.5 µM 1:4 68.4 ± 3.1

4 µM 2:1 82.4 ± 2.3

strategy B (custom patterning) 0.5 µM saturated 76.4 ± 4.1

2 µM saturated 87.5 ± 3.5

4 µM saturated 85.2 ± 3.6

Blotting voltage 100 V/cm and 200 µm long membrane. b Concentration in polyacrylamide precursor solution.

Figure 4. Capture efficiency of the blotting membrane is influenced by the applied electric field strength and membrane axial length. Two blotting membrane lengths (O, 200 µm; b, 500 µm) were considered for a range of applied blotting electric field strengths. Inset images show inverted grayscale CCD images of blotting membrane after capture of R-actinin at a concentration of 0.1 µM.

Two important factors that impact the residence time of the analyte in the blotting membrane include the axial length of the blotting membrane and the migration speed of species through the blotting membrane. The slit size on the photomask, the distance between the mask and channel surface, and the exposure duration determine the final length of the photopolymerized region.33 Membranes as short as 20 µm and as long as 4 mm have been fabricated using this approach (data not shown). Figure 4 shows results from characterization of two blotting membrane geometries having different axial lengths (200 and 500 µm), both being compatible with the channel layouts used in this study. Low electric field strength operation (100 V/cm), a longer membrane (>200 µm) was employed so as to yield sufficient capture. A higher % T PA gel (>8% T) can also be used to yield a blotting membrane with smaller pore-size. In the case of the longer 500 µm blotting membranes, a ∼100% capture efficiency was observed over the range of electric field studied (25-275 V/cm). While the optimal conditions required for maximizing capture % are dependent on several factors (i.e., gel pore size, (33) Throckmorton, D. J.; Shepodd, T. J.; Singh, A. K. Anal. Chem. 2002, 74, 784–789. (34) Duchesne, L.; Fernig, D. G. Anal. Biochem. 2007, 362, 287–289.

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Figure 5. Blotted proteins show a dose response behavior allowing development of quantitative immunoblotting. (a) Inverted grayscale CCD images show fluorescence of proteins bound to in-channel antibody-functionalized blotting membranes. E ) 50 V/cm. (b) Calibration curve of R-actinin shows enhanced detection sensitivity on the blotting membrane (b), as compared to signal without the blotting membrane (O). Curves were fit by the nonlinear least-squares method.

the affinity between the migrating species and the immobilized antibodies, and the blotting antibody immobilization density), a blotting membrane length of 200 µm was sufficient for efficient capture under operating conditions utilized in this work. Toward Protein Quantitation on the Blotting Membrane. To relate the concentration of target protein in the sample to the fluorescence signal generated at the blotting membrane, a calibration curve was obtained through analysis of a protein dilution series (Figure 5). Dose-response behavior was obtained by fluorescence imaging of blotting membranes during analysis of BSA (against streptavidin-functionalized blotting membranes), R-actinin (against anti-actinin blotting membranes), and PSA (against anti-PSA blotting membranes). Free dye was spiked into the dilution series at a set concentration

and used to normalize the fluorescence signal detected on and off the blotting membrane. To avoid sample carry-over, new immunoblot chips were used for each new sample. Figure 5a shows CCD images of membranes blotted with selected concentrations of BSA, R-actinin, and PSA. As is visually reported in the images, fluorescence signal from blotting membranes for both R-actinin and PSA showed a dosedependent response, as higher concentrations of protein were introduced. Note that the increase in intensity is more subtle for PSA than the response for R-actinin, an observation that may be attributable to the variation in response inherent to species-specific binding affinities. A 1.8 pg mass of PSA was readily detectable in the CCD images. For the concentration of R-actinin considered, blotting membranes successfully bound a substantial mass of analyte (19.8 pg mass in the peak). No such signal dependence on concentration was observed for the negative control, BSA. Figure 5b reports the full dose-response behavior of anti-actinin blotting membranes. A linear response in fluorescence signal was observed over a 101-103 nM concentration range. Interestingly, target protein enrichment occurred on the blotting membrane and thus yielded ∼5fold enhanced detection sensitivity (Figure 5b) above corresponding protein solutions detected without the blotting membrane. A similar effect has been observed in protein microarrays.35 The lowest R-actinin sample mass captured and detected on the blotting membrane was ∼0.05 pg. Our results indicate the potential to improve detection limits over slab-gel immunoblotting36 through lossless manipulation of minute sample masses (picograms), as is especially relevant to single cell studies.37 Alternate Strategy for Antibody Patterning of Blotting Membrane. The development of the custom patterning strategy (described in Materials and Methods) allows end-user flexibility in the identity of biotinylated blotting reagents (e.g., antibodies, aptamers, Fab fragments), even after device fabrication, storage, and transport. The flexibility of the custom patterning strategy was demonstrated in Figure 6. Both CCD images and electropherograms showed the selective recognition of the target protein according to the antibodies patterned by an end-user. Additionally, the custom patterning strategy typically required 10 µL of ∼1 µM biotinylated antibody reagent in the patterning well. On the basis of loading estimates, ∼1 µg of biotinylated antibody reagent was consumed during the immobilization process. In comparison, conventional slab-gel Western blotting commonly requires ∼8 µg of antibody reagent for blotting.7 CONCLUSIONS We introduce a new approach to immunoblotting that takes advantage of microfluidic technology to simplify integration of multistep assays. Here we introduce postseparation (i.e., “downstream”) functionality that allows reporting of analyte apparent mobility as well as identification through a down(35) Rubina, A. Y.; Pan’kov, A. V.; Dementieva, E. I.; Pen’kov, D. N.; Butygin, A. V.; Vasiliskov, V. A.; Chudinov, A. V.; Mikheikin, A. L.; Mikhailovich, V. M.; Mirzabekov, A. D. Anal. Biochem. 2004, 325, 92–106. (36) Delaive, E.; Arnould, T.; Raes, M.; Renard, P. J. Immunol. Methods 2008, 334, 51–58. (37) Dishinger, J. F.; Reid, K. R.; Kennedy, R. T. Anal. Chem. 2009, 81, 3119– 3127.

Figure 6. Custom patterned blotting membranes show capture of specific target proteins after native PAGE. Inverted grayscale CCD images and companion electropherograms were collected both upstream and downstream of membranes. R-actinin (a) and PSA (b) were selectively captured by corresponding antibodies. E ) 80 V/cm, [PSA] ) ∼20 nΜ, [R-actinin] ) ∼10 nΜ.

stream immunoblotting assay. The seamless microfluidic integration of multiple steps (separation, transfer, blotting) yields a streamlined workflow with potential for programmable, “hands free” operation. Initial characterization of the PAGE in situ immunoblotting assay indicates that the scheme is a highly specific and sensitive approach that yields no discernible sample loss, suggesting a well-suited format for automated, quantitative microfluidic immunoblotting. Successful assessment of R-actinin in a multiprotein sample further suggests that the native PAGE in situ immunoblot provides the capability to assay large molecular weight species, a challenge in conventional membrane-based blotting. A flexible custom patterning strategy was demonstrated for postfunctionalization of in-chip blotting membranes. The custom patterning strategy yielded substantially greater versatility than the prepatterning strategy for customization of the immunoblot by an end-user. Currently, optimization of the blotting membrane function, enhanced applicability through integration of the immunoblot with protein sizing (sodium dodecyl sulfate PAGE), and analysis of complex protein samples relevant to both basic science and clinical questions are underway. We see great potential to extend the basic assay developed and presented here to panels of unique blotted proteins, as well as further improvements in detection sensitivity through on-chip protein enrichment. Additional design goals include robust quantitation (immunoblotto-immunoblot reproducibility in quantitation) not readily possible without fine control of protein transfer. While further optimization is underway, the approach demonstrated here holds substantial promise as the basis for a suite of automated Analytical Chemistry, Vol. 81, No. 19, October 1, 2009

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immunoblotting technologies, including microfluidic Western blotting. ACKNOWLEDGMENT The authors greatly appreciate equipment and glass device fabrication support from Caliper Life Sciences. The authors thank Chenlu Hou and Akwasi Apori at UC Berkeley for their assistance. The authors also thank the California Institute for Quantitative Biosciences (QB3) at UC Berkeley and UC San Francisco, The Rogers Family Foundation, and the National Science Foundation

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Center for Integrated Nanomechanical Systems (COINS) for generous financial support. A.E.H. also thanks the UC Berkeley Regents’ Junior Faculty Fellowship and the Hellman Family Faculty Fund Award.

Received for review June 25, 2009. Accepted August 18, 2009. AC901392U